This document discusses bioremediation techniques for cleaning up contamination from hydrocarbons like crude oil. It explains that most hydrocarbons can be degraded by microorganisms under both aerobic and anaerobic conditions. Lighter and simpler hydrocarbons are more readily biodegraded, while heavier compounds like those found in crude oil can persist in the environment. The document outlines some of the physical, chemical, and biological processes that affect the behavior and degradation of hydrocarbon contamination in soil and groundwater.

1. D R . A N A N D M A D H A V A N
S C H O O L O F E N V I R O N M E N T A L S T U D I E S
C O C H I N U N I V E R S I T Y O F S C I E N C E A N D
T E C H N O L O G Y
K O C H I – 2 2 . K E R A L A , I N D I A
Bioremediation: Techniques
for Cleaning up a mess

2. What is Bioremediation ?
Bioremediation is the transformation or degradation
of contaminants into non-hazardous or less hazardous
Chemicals
 compost piles existed as far back as 6000 BC.
 In 1891 the first biological sewage treatment plant
was created in Sussex, UK.
However, the word “bioremediation” did not appear in
peer-reviewed scientific literature until 1987.

3. Why consider bioremediation ?
 isolation by capping with man made or natural
materials.
 excavation and transport to landfill.
 The toxic materials from these “dig and dump” sites
have begun to leak into water sources and into areas
that sustain human life.
 This problem has led to modern-day bioremediation.

4. Pollution and Pollution control
Pollution is one of the most prevailing topics in today's
environmental discussions and a subject of continuous
legislation
The awakening to existence of chemical pollution…
 “The silent spring” (1962) – Book by Rachel Carson
 Love Canal case in USA (1978)
 Bhopal disaster of India (1984)
 Chernobyl nuclear reactor (1986)
 Exxon Valdez oil-spill (1989)

5. Why is bioremediation a favourable alternative to
current practices?
 destruction of contaminants
 potentially quicker cleanup times
 potentially lower costs
 minimum disruption to ongoing operations on the
site
 reduced or eliminated transport costs
 reduced future liability
 highly versatile

6. Limitations of bioremediation
Bioremediation can only be utilised to treat soil/water
contaminated with organic compounds which are
susceptible to microbial degradation.
Other limitations are…
 Pre-treatment Assessment
 Time scale
 Uncertainty

7. Remediation Technique Approximate Costs Range
 Incineration £100 - £400 /m3
 Landfilling £30 - £75 /m3
 Capping £20 - £180 /m3
 Soil washing £15 - £40 /m3
 Stabilisation £10 - £100 /m3
 Bioremediation £1 - £150 /m3
(Adapted from Contaminated Land Remediation: A Review of Biological Technology 2000)

8. Successfully biodegraded materials
Diesel
Jet Fuel
Paraffin
Ammonia
Crude Oil
Lube Oil
Petrol
Fuel Oils
Alcohols
Acids
Aldehydes
Ketones
Phenols
Chlorophenols
Creosote
Explosives*
PCBs*
PAHs*
Tars*
Chlorinated* Solvents
Pesticides*
Herbicides*
Cyanides*
* = biodegraded only under certain circumstances

9. Materials which have not been
successfully biodegraded
 Bitumen
 Asphalt
 Inorganic Acids
 Asbestos
 Metals*
* = non-biodegradable

10. classifications of bioremediation:
 Biotransformation - the alteration of
contaminant molecules into less or nonhazardous
molecules
 Biodegradation - the breakdown of organic
substances in smaller organic or inorganic molecules
 Mineralization - is the complete biodegradation of
organic materials into inorganic constituents such as
CO2 or H2O

11. Novel Bioremediation Techniques

12. In-Situ Ex-Situ
 Time relatively
unrestricted
 Widespread
contamination
 Low to medium
concentration
 Deep within the site
 Less than a year
 Localised
contamination
 Medium to high
concentration
 Relatively near surface
Why In-Situ or Ex-Situ ?

13. Bioremediation terminology
 Biodegradation
 in-situ bioremediation
 ex-situ bioremediation
 Biostimulation
 bioaugmentation
 natural attenuation
 Bioavailability
 abiotic losses
 biotic losses

14. Factors which influence bioremediation
 presence of a suitable micro flora
 chemical nature of pollutants
 temperature
 oxygen level
 moisture level
 presence of co-occurring contaminants
 soil type
 nutrient levels

15. Bioremediation mechanism
 Microorganisms destroy organic contaminants in the
course of using the chemicals for their own growth
and reproduction
 Organic chemicals provide:
carbon, source of cell building material
electrons, source of energy
 Cells catalyze oxidation of organic chemicals (electron
donors), causing transfer of electrons from organic
chemicals to some electron acceptor

17. Microbial Metabolism
 Need nitrogen, phosphorus, sulfur, and a variety of
trace nutrients other than carbon
 Carbon is often the limiting factor for microbial
growth in most natural systems
 Acclimatization period - a period during which no
degradation of chemical is evident; also known as
adaptation or lag period
 Length of acclimatization period varies from less than
1 h to many months
 Acclimatization of a microbial population to one
substrate frequently results in the simultaneous
acclimatization to some structurally related molecules

18. Metabolism Modes
 Aerobic: transformations occur in the
presence of molecular oxygen (as electron
acceptor), known as aerobic respiration
 Anaerobic: reactions occur only in the
absence of molecular oxygen, subdivided
into:
– Anaerobic respiration
– Fermentation
– Methane fermentation

19. Metabolism Modes
 Anaerobic respiration
 – Nitrate as an electron acceptor - denitrifying and nitratereducing
 organisms
 – Sulfate and thiosulfate as electron acceptors - by sulfatereducing
 organisms
 – CO2 as an electron acceptor, by methanogenic organisms
 – Chlorinated organic compounds as electron acceptors
 • Fermentation - organic compounds serve as both
 electron donors and electron acceptors
 • Methane fermentation - consecutive biochemical
 breakdown of organic compounds to CH4 and CO2

20. Metabolism Modes
 Cometabolism - transformation of an organic
 compound by a microorganism that is unable
 to use the substrate as a source of energy
 • Metabolites or transformation products from
 cometabolism by one organism can typically
 be used as an energy source by another
 • Preferential degradation: higher energy
 yielding compounds degraded first, e.g., in a
 petroleum spill under aerobic conditions,
 benzene → naphthalene →chrysene

21. Microbial Reactions and Pathways
 Dechlorination - a chlorine atom is replaced with a
 hydrogen atom
 • Hydrolysis - a cleavage of an organic molecule with
 the addition of water
 • Cleavage - an organic compound is split or a terminal
 carbon is cleaved off an organic chain
 • Oxidation - breakdown of organic compounds using
 nucleophilic form of oxygen (H2O, OH-, etc); releases
 electrons
 • Reduction - breakdown of organic compounds using
 electrophilic form of hydrogen (H+); takes electrons

22. Hydrocarbons
 Where does hydrocarbon contamination
come from?
 Why are hydrocarbons toxic?

23.  Hydrocarbons can be introduced into the
environment via incomplete combustion of fossil
fuels, accidental discharge during transport, the
disposal of petroleum products and other organic
wastes, incineration of refuse and wastes, and
various industrial processes. They can also be
produced as a result of natural processes including
forest fires and volcanic eruptions. Certain
hydrocarbons known as polycyclic aromatic
hydrocarbons (PAHs) are found in soil at wood
preservation plants and gas manufacturing facilities.

24.  The inertness of high molecular weight hydrocarbons
coupled with low solubility in water and strong
lipophilic character lead to high accumulation levels
and persistence. Some hydrocarbons (such as
benzene) have been shown to be carcinogenic. In the
case of large industrial oil spills, the oil can actually
suffocate living organisms and destroy entire
ecosystems. Excess hydrocarbons in aqueous
environments can deplete the supply of oxygenating
molecules, leading to anoxic conditions.

25. Why use bioremediation?
 Although they can be eliminated abiotically through
photooxidation, PAHs with more than three rings are
more difficult to remove. High-molecular weight
PAHs have a low bioavailability due to their strong
adsorption onto the soil organic matter, high
resonance energy, and toxicity. Numerous
microorganisms – bacteria, algae, and fungi – are
capable of metabolizing hydrocarbons.

26. Hydrocarbons: Biological Processes
 Most crude oils contain hydrocarbons ranging in size
from methane to molecules with hundreds of
carbons. When crude oils reach the surface
environment, they are biodegraded, at least under
aerobic conditions.

27. Basics of Crude Oil
Crude oils are complex mixtures containing many
different hydrocarbon compounds that vary in
appearance and composition from one oil field to
another. Crude oils range in consistency from
water to tar-like solids, and in colour from clear to
black. An "average" crude oil contains about 84%
carbon, 14% hydrogen, 1%-3% sulphur, and less
than 1% each of nitrogen, oxygen, metals, and salts.
Crude oils are generally classified as paraffinic,
naphthenic, or aromatic, based on the
predominant proportion of similar hydrocarbon
molecules.

28. TYPICAL APPROXIMATE CHARACTERISTICS AND
PROPERTIES AND GASOLINE POTENTIAL OF VARIOUS CRUDES
(Representative average numbers)
Crude source Paraffins
(% vol)
Aromatics
(% vol)
Naphthenes
(% vol)
Sulphur
(% wt)
Nigerian - Light 37 9 54 0.2
Saudi - Light 63 19 18 2
Saudi - Heavy 60 15 25 2.1
Venezuela - Heavy 35 12 53 2.3
Venezuela - Light 52 14 34 1.5
USA - Midcont. Sweet - - - 0.4
USA - W. Texas Sour 46 22 32 1.9
North Sea - Brent 50 16 34 0.4

29. IMPACT OF CRUDE OIL ON THE
ENVIRONMENT
 Toxic to humans/fauna/flora by ingestion, inhalation,
and transport across membrane structures;
 Groundwater contamination ;
 Physical impact, e.g. soil structure denaturisation, water
ingress prevention, increased toxicity levels;
 Physical impact on biota, e.g. coating of avian plumage,
blockage of invertebrate respiratory and feeding
mechanisms, blockage of sunlight on water surface;
 Prevention of use of amenities;
 Consequential economic impacts;
 Consequential social impacts.

30. Petroleum hydrocarbons
 Main components are alkanes (straight, branched
and cyclic), alkenes, mono-aromatics (benzene,
toluene, ethylbenzene and xylene (BTEX)) and
poly-aromatic compounds
 Alkanes, alkylaromatics and aromatics in the C22
range have low toxicity, but their stability at 35 C
makes them difficult to biodegrade
 Microorganisms can degrade them, but it will take
time
 Condensed aromatics and cycloalkane systems are
very resistant to biodegradation Double bonds add greater
resistance to complete degradation

31. Relative biodegradability
 Simple hydrocarbons and petroleum fuels
 degradability decreases as molecular weight and degree of
branching increase
 Aromatic hydrocarbons one or two ring compounds degrade readily,
higher molecular weight compounds less readily
 Alcohols, esters
 Nitrobenzenes and ethers degrade slowly
 Chlorinated hydrocarbons decreasing degradability within increasing
chlorine substitution – highly chlorinated compounds like PCBs and
chlorinated solvents do not appreciably degrade aerobically
 Pesticides are not readily degraded

32. Factors Affecting Hydrocarbon
Concentration and Mobility
The persistence of the contaminant in the
environment is dependent upon the initial
composition and concentration of the hydrocarbon
contamination and other environmental parameters
in processes known collectively as Natural
Attenuation. Natural Attenuation involves the
physical processes, the biological action
(biodegradation), and any combination of these
processes.

33. Physical Degradation (or conversion).
 Volatilisation and dissolution tends to remove low molecular weight
aromatics and aliphatics
 Hydrodynamic dispersion - relates to aqueous redistribution of
contaminants
 Dissolution is very important for soluble contaminants which
breakdown in the presence of water (hydrolysis)
 Sorption - reduction of contaminant availability and mobility due to
chemical and physical binding within the soil environment. A given
volume of strata can adsorb a given amount of contaminants; hence with
very concentrated hydrocarbon spills this process can be overwhelmed as
the ground exceeds its "sorption capacity"
 Dilution - reduction of concentration although increased mobility
 Abiotic degradation or chemical transformation involves the
breakdown of contaminant molecules by physiochemical processes (e.g.
cation exchange)

34. Biodegradation
Initially, biodegradation favours the removal of n-
alkanes, low molecular weight cycloalkanes and
light aromatics since they are more
chemically/physically susceptible to metabolism by
soil organisms. The action of biodegradation is
more pronounced at the periphery of contaminant
plumes where sufficient Redox (electron acceptor)
compounds (oxygen, nitrate, iron, sulphate and
carbon dioxide) are present. The more
concentrated a hydrocarbon plume the less the
impact of biodegradation.

35. Hydrocarbon Behaviour in the
Sub-Surface
Hydrocarbons that escape into the environment
behave differently depending upon their chemical
constituents and the environment they encounter.
Residual/Adsorbed Hydrocarbons
Free product migrates through strata by
‘smearing’, leaving product in the pore spaces,
which frequently gets either trapped or binds to
the surface of the strata it passes through. This can
act a source of continued contamination in the
event of groundwater level fluctuations, or rainfall
percolation.

36. Volatilised Constituents
A proportion of the more volatile fractions of any
hydrocarbon escape may migrate away in the gas phase, and
even reach to the surface as part of a vapour plume.
Hydrocarbons and Water
When free product encounters water, a proportion of the
hydrocarbons will, after a while, dissolve, float or sink,
dependent upon factors such as solubility and the
hydrocarbon type.

37. Dissolved Phase
Hydrocarbons with a high relative solubility are likely to
dissolve in the water and be more mobile than other, heavier
hydrocarbons. Parameters of interest are the solubility and
partition coefficient (i.e. a measure of how readily and to
what extent hydrocarbons will dissolve in water).
LNAPLs - Light Non-Aqueous Phase Liquids
These refer to free phase hydrocarbons that float on water.
Although less mobile than the dissolved phase hydrocarbons,
they can act as a further source of mobile hydrocarbon in a
contaminant plume.

38. DNAPLS - Dense Non-Aqueous Phase Liquids
These represent heavier compounds that readily
sink in water and are the least mobile of all the
hydrocarbon groups (e.g. tar, heavy oils, etc). They
can break down over time to sustain an elevated
concentration of the lighter more mobile
hydrocarbon fractions. They are very persistent in
the environment, bioaccumulate in living tissue,
and frequently contain toxic compounds.

39. Hydrocarbon Vapours
Many hydrocarbon mixtures in the aqueous environment can
still contain volatile fractions, which can return to the gas
phase at a distance from the source.
Metals
These can occur as naturally occurring components of crude
(e.g. vanadium, nickel).

40. Some examples of hydrocarbons found in crude
oil

41. Degradation of Aliphatic Hydrocarbons
Generally an aerobic process
• As high as 20% of all soil microbes (bacteria, fungi
and yeast) are capable of degrading aliphatic
hydrocarbons
• Most common pathway of alkane degradation is
oxidation at the terminal methyl group; alkane →
alcohol → fatty acid → ketone → CO2 and H2O;
short chain hydrocarbons (except methane) more
difficult to degrade
• Unsaturated straight-chain hydrocarbons generally
less readily degraded than saturated ones
• Hydrocarbons w/ branch chains and cyclic aliphatic
hydrocarbons less susceptible to biodegradation

42. Initial steps in the biodegradation of linear and
cyclic alkanes:

43. Degradation of Aromatic Hydrocarbons
Microorganisms capable of aerobically
metabolizing single-ring aromatic hydrocarbons
ubiquitous in the subsurface
• PAHs with two or three rings such as
naphthalene, anthracene, and phenanthrene are
degraded at reasonable rates when O2 is present
• PAHs with four rings such as chrysene, pyrene,
and pentacyclic compounds are highly
persistent and are considered recalcitrant

44. Oxidation of Benzene to catechol

45. Ortho-cleavage pathway for catabolism
of catechol

46. Single-Ring Aromatics

47. PAHs with 2-3 Rings

48. PAHs with 4-5 Rings

50. Degradation of Chlorinated Aliphatic
Hydrocarbons (CAHs)
Can occur both chemically (abiotic) and
biologically (biotic)
• Generally transformed only partially by
microbial processes
• Only the less chlorinated one- and two-carbon
compounds might be used as primary substrates
for energy and growth, and organisms capable
of doing this not widespread in the environment
• Microbial transformation of most CAHs
depends upon cometabolism

51. Halogenated Organic Solvents:

52. Anaerobic Degradation CAHs

53. Reductive dechlorination of carbon tetrachloride
and tetrachloroethylene

54. Aerobic Oxidation of
Tetrachloroethylene

62. Where/How Can Bioremediation Fail?
1. Absence of contaminant – degrader population
2. Microbes, contaminants, nutrients not co-located
3. Other organisms out compete the contaminant-degraders Activity or
numbers can not be adequately increased to achieve desired removal
rate.
4. Sick contaminant – degrader population
• substrate: alternative substrate ratio too high or too low
• mutation & selection favor poorly performing strains
• crossed signals (microbes communicate w/chemical signals)
• byproducts toxic/inhibitory (transfer Rates too high)
• non-target organisms inhibitory
* Functional stability is not guaranteed even if
contaminant-degrader population is large

63. A Few examples of failed bioremediation attempts (which
can be costly):
 Inoculation of soil with aliphatic hydrocarbon
degrading bacteria did not enhance degradation of
fuel oil
 A Pseudomonas sp. shown in lab cultures to degrade
1,4-dichlorophenol failed to degrade the compound
when added to surface soils
(source: Watwood, Maribeth 2003)

64. Site Specificity:
 Each new site represents a different set of
conditions, and what works in the lab may not
translate to the field.
 Laboratory experiments can be carefully controlled,
but these optimal conditions may not be possible at
the site and may misrepresent possibility for success
in the field

65. Fears:
 introducing “foreign” microorganisms to field sites
could have unforeseen consequences on the
ecosystem
 Concerns about genetically modified organisms
used for bioremediation are basically the same as
concerns about using GMO’s in general. There is
worry about
 Horizontal gene transfer
 Creation of new pathogens
 Possibility of mutations that allow the organism to
become invasive.

66. Limitations of Bioremediation
Failures:
Most failures at bioremediation are due to failure
of introduced organisms to thrive in the natural
environment or a failure to access the
contaminant. This could be due to:
Lack of nutrients
Predation or parasitism
Competition
Immobility of introduced bacteria
Contaminant concentrations below threshold for organism
survival
Organisms may feed on alternative substrates

67. Alternatives
 Hydrocarbons
Burning off oil spills at sea.
Has several drawbacks:
the ignition of the oil;
maintaining combustion of the slick;
the generation of large quantities of smoke;
the formation and possible sinking of extremely
viscous and dense residues;
and safety concerns

68. Benefits of Bioremediation

69.  Cost effective, because contaminants can be
treated in situ.
 Capitalizes on a natural processes that occurs
anyway.
 Can be used to treat dispersed contaminants in the
environment.
 Minimizes disturbance to the environment and
danger to workers.
 Can mineralize contaminants completely and
eliminate the need for disposal.

70. Acid mine drainage

71. Metals
 Metal contamination:
A variety of metal contaminents exist in
groundwater, surface water, and soils resulting from
industrial and agricultural activity. Toxic metals such
as lead, mercury, cadmium, arsenic, chromium, and
uranium can cause damage to human health and the
environment.

72. How does metal contamination occur?
 Perhaps the most prevalent and problematic form
of metal pollution is acid mine drainage. This
occurs when the mining of coal and metal ores
exposes metals and radionuclides to the
atmosphere allowing them to be oxidized by
certain bacteria (Thiobacillus ferrooxidans). For
example pyrite (often exposed in coal mining) can
be oxidized to iron hydroxide and sulfuric acid:
FeS2 + 15/4 O2 + 7/2 H2O = Fe(OH)3 + 2H+ +
2HSO4-

73. Why use bioremediation?
 Unlike organic compounds, metals cannot be broken
down into non-toxic components. However,
biological orgainisms can naturally reduce their
toxicity through processes such as chelation and
precipitation.

74.  Chelates are used by many organisms including
plants and bacteria to aid the absorption and
transportation of essential metal nutrients. However,
their binding properties can often be used to stabilize
toxic metals as well. Thus chelates are critical
compounds for bioremediation, especially in
phytoremediation.

75.  Metallothionens:

76.  Phytochelatin binded to Cd:

77. Metal Precipitation
 Metals can be removed from acid mine effluents as
solid precipitates by anaerobic bacteria. Through
redox reactions the bacteria reduce the oxidation
state of the metal, which usually causes it to form a
harmless solid precipitate. For instance, iron
reducing bacteria like Geobacter who normally
gain energy by reducing Fe (III) to Fe (II) can
reduce U (VI) to U (IV) instead. The reduced form
of the metal then forms a non-toxic solid
precipitate.

78.  Sulfur reducing bacteria such as Desulfovibrio can also be
used for bioremediation, though the chemistry requires an
extra step.
 First the bacteria reduces the sulfate producing hydrogen
sulfide:
 SO4-2 + 2CH2O H+ = H2S + H2O +CO2
 Hydrogen sulfide (H2S) then reacts with metals to form a
sulfide that preciptates out of the effluent. Additionally,
some bicarbonate (HCO3-) produced along with the CO2 in
the sulfur redox reaction that acts to neutralize the acid in
the effluent. With the acidity reduced and the metals now
existing as a harmless precipitate the effluent is effectively
remediated.

79. In Situ Bioremediation
 Intrinsic Bioremediation
 Enhanced bioremediation

80. Intrinsic In Situ Bioremediation
 Intrinsic bioremediation relies on natural
processes to degrade contaminants without
altering current conditions or adding amendments.
 “biodegradation, dispersion, dilution, sorption,
volatilization, radioactive decay, and chemical or
biological stabilization, transformation or
destruction of contaminants” (NRC, 2000; EPA,
1999).

81. Enhanced In Situ Bioremediation
Enhanced bioremediation can be applied to ground
water,vadose zone soils, or, more rarely, aquatic
sediments. Additives such as oxygen (or other
electron acceptors), nutrients, biodegradable
carbonaceous substrates, bulking agents, and/or
moisture are added to enhance the activity of
naturally occurring or indigenous microbial
populations (FRTR, 2003).

82. Bioremediation technologies for soil
 Composting – addition of moisture and nutrients,
regular mixing for aeration
 Biopiles – ex-situ aeration of soil
 Land treatment – application of organic materials
to natural soils followed by irrigation and tilling
 Bioventing – in-situ aeration of soil

83. Composting
Source: U.S.AEC, 2000. Windrow
Composting of Explosives-Contaminated
Soil. U.S. Army Environmental Center.
(http://aec.army.mil/prod/usaec/et/resto
windrow.htm)

84. Source: USAEC, 2000. Biopiles
of POL Contaminated Soils. U.S.
Army Engineer Environmental
Center.
(http://aec.army.mil/prod/usaec/et
/restor/pol01.htm)

85. Biopile
Source: Environmental
Protection Agency, Tech Trends
newsletter, June 2001.
http://www.epa.gov/swertio1/do
wnload/newsltrs/tt0601.pdf.
Accessed May 11, 2004.

86. Requirements for soil bioremediation

87. Vadose Zone Soil Remediation
 The primary in situ biological technology applicable
to the unsaturated zone is bioventing, which is
categorized as either aerobic, cometabolic, or
anaerobic depending on the amendments used.

88. Aerobic Bioventing
 Bioventing has a robust track record in treating
aerobically degradable contaminants, such as fuels.
In aerobic bioventing, contaminated unsaturated
soils with low oxygen concentrations are treated by
supplying oxygen to facilitate aerobic microbial
biodegradation.
 Bioventing is designed primarily to treat
aerobically biodegradable contaminants, such as
non-chlorinated VOCs and SVOCs (e.g., petroleum
hydrocarbons), that are located in the vadose zone
or capillary fringe (EPA, 2000; FRTR, 2003).

89. Engineered Bioremediation of Unsaturated Zone
(Bioventing)

90. Limitations of Bioventing
 One set of bioventing limitations involves the ability to
deliver oxygen to the contaminated soil. For example, soils
with extremely high moisture content may be difficult to
biovent because of reduced soil gas permeability.
 Similarly, low-permeability soils also may pose some
difficulties for bioventing because of a limited ability to
distribute air through the subsurface.
 Additionally, sites with shallow contamination can pose a
challenge to bioventing because of the difficulty in
developing a system design that can minimize
environmental release and achieve sufficient aeration.

91. Cometabolic Bioventing
 Cometabolic bioventing has been used at a few
sites to treat chlorinated solvents such as TCE,
trichloroethane (TCA), and dichloroethene (DCE).
 Cometabolic bioventing exploits competitive
reactions mediated by monooxygenase enzymes
(EPA, 2000).
 Thus, by supplying an appropriate organic
substrate and air, cometabolic bioventing can elicit
the production of monooxygenases, which
consume the organic substrate and facilitate
contaminant degradation (AFCEE, 1996; EPA,
1998a).

92. limitations
 design of cometabolic bioventing systems are
dependent on many factors including soil gas
permeability, organic substrate concentration, type
of organic substrate selected, and oxygen supply and
radius of influence.

93. Anaerobic Bioventing
 Anaerobic bioventing uses the same type of gas
delivery system as the other bioventing
technologies, but injects nitrogen and an electron
donor, instead of air, to establish reductive
anaerobic conditions.
 The nitrogen displaces the soil oxygen, and small
amounts of an electron donor gas (such as
hydrogen and carbon dioxide) produce reducing
conditions in the subsurface, thereby facilitating
microbial dechlorination.

94. Anaerobic Bioventing
 This process may be useful in treating highly
chlorinated compounds such as tetrachloroethene
(PCE), TCE, RDX, pentachlorophenol, and pesticides
such as lindane and dichloro-diphenyl-trichloro-
ethane (DDT).

95. Surficial Soil Remediation
 If contamination is shallow, soil may be treated in
place using techniques similar to land treatment or
composting. Variations of these technologies involve
tilling shallow soils and adding amendments to
improve aeration and bioremediation.

96. Ground Water and Saturated Soil Remediation
 In situ bioremediation techniques applicable to
ground water and saturated soil include
dechlorination using anaerobic reducing conditions,
enhanced aerobic treatment, biological reactive
barriers that create active remediation zones, and
bioslurping/biosparging techniques that promote
aerobic degradation.

97. common organic contaminants
Halogenated
Alkanes (CT, TCA, DCA, Freon 11, Freon 113)
Alkenes (PCE, TCE, DCE,VC)
Phenols, Chlorobenzenes, PCBs, Dioxins, Furans
Non-Halogenated
Acetone, Acrolein
Alcohols (n-Butyl alcohol, isobutanol, methanol)
Cyclohexane, Phthalates, Benzoic acid, PAHs
BTEX ,Methyl ethyl ketone (MEK) , Petroleum
hydrocarbons

98. common organic contaminants
Pesticides
BHC, DDD, DDE, DDT, Endrin, Ethion, Malathion,
Toxaphene
Explosives
TNT, TNB, DNB, RDX, HMX, Nitroglycerin,
Nitrocellulose

99. common organic contaminants
Metals
Ag, Al, As, Be, Cd, CN, Cr, Cu, Hg, Fe, Ni, Pb, Sb, Se,
Zn
Radioactive Elements
- Low level radioactive waste
- Transuranic waste including:
Pu-238, Pu-239, Ra-224, Ra-226
Th-228, Th-230, Th-232
U-234, U-235, U-238

103. overview: in situ ground water bioremediation
Technology Definition: Use of indigenous
subsurface microorganisms to transform, destroy or
immobilize contaminants in the saturated zone in
place.
Goal: Detoxification of the parent compound(s) and
conversion to products that are no longer hazardous
to human health and the environment.

104. Feasibility/Effectiveness is a function of:
– Contaminant type & state
(biodegradability/availability)
– Environmental factors (affect microbial activity and
rate)
– Site conditions (geological & chemical)

105. Four metabolic processes:
Aerobic respiration
Anaerobic respiration
Fermentation: External electron acceptors are not
required; contaminant serves as both electron
donor and electron acceptor.
Cometabolism: Simultaneous metabolism of two
compounds in which the degradation of the
second compound (secondary substrate) depends
on the presence of the first compound (primary
substrate).

106. Contaminant Biodegradability: The inherent
property of an organic contaminant to be broken
down biologically under a set of specified
environmental/microbial conditions
– Depends on physical/chemical properties (e.g.,
water solubility, octanol/water partition coefficient)
Biodegradability Assessment:
– Literature
– Experimental
– Structure-activity relationships

107. Environmental Factors:
– Temperature
– pH
– Nutrients (organic & inorganic; availability)
– Electron acceptor(s)
– Redox potential
– Water activity (or potential)
– Osmotic pressure
– Type and concentration of contaminant(s)

108. Engineered Bioremediation:
 Enhancement/acceleration of microbial activities
Using engineered procedures to isolate and control the
contaminated site.
 Can be combined with other remedial technologies:
»Saturated zone (e.g., air sparging)
»Vadose zone (e.g., soil vapor extraction,
bioventing)

109. Aerobic Treatment
 Similar to bioventing, enhanced in situ aerobic ground
water bioremediation processes are used in situations
where aerobically degradable contaminants, such as fuels,
are present in anaerobic portions of an aquifer.
 air or other oxygen sources are injected into the aquifer
near the contamination
 the oxygenated water migrates through the zone of
contamination, the indigenous bacteria are able to degrade
the contaminants (EPA, 1998a; EPA, 2000).
 Aerobic treatment may also be used to directly or
cometabolically degrade lightly chlorinated species, such as
DCE or VC.

110. Engineered Bioremediation of Unsaturated and
Saturated Zones

111. Biosparging and Bioslurping
 Biosparging (similar to air sparging) involves the
injection of a gas (usually air or oxygen) and
occasionally gas-phase nutrients, under pressure,
into the saturated zone to promote aerobic
biodegradation (GWRTAC, 1996).

112. Biosparging

113. Bioslurping

114. technical process -- five steps/phases
1. Site investigation
2. Treatability studies
3. Recovery of free product and removal of the
contamination source
4. Design and implementation of the in situ
bioremediation system
5. Monitoring and performance evaluation of the in
situ bioremediation system

115. technical process
1. Site investigation
 Aquifer Characterization
 Biological Characterization
 Contaminant Characterization

116. site characteristics favoring in situ
bioremediation

117. 2. Treatability studies (Simulation of field conditions)
- Assessment of bioremediation potential based on
site-specific variables
- Estimation of the rate and extent of
biotransformation
- Determination of nutrient requirements
3. Recovery of free product and removal of the
contamination source

118. 4. Design and implementation of the in situ
bioremediation system
- Plume control (Hydraulic control or slurry barriers)
- Process enhancement by addition of: nutrients, oxygen
or other electron acceptor(s), and/or amendments
(e.g., surfactants)
- Establish “standards” that will be used to evaluate
process performance
- Design should be flexible/adjustable based on
operational data

119. 5. Monitoring and performance evaluation of the in
situ bioremediation system
Three types of evidence:
a. Documented loss of contaminant(s) from the
site
b. Laboratory assays to document the biotransformation
potential of site microorganisms under site conditions
c. Evidence that the biotransformation potential is
realized in the filed

120. advantages of engineered in situ
bioremediation
1. Remediates contaminants dissolved in ground water as well as
those sorbed or trapped within the geologic materials.
2. Application involves equipment that is widely available and easy to
install.
3. Creates minimal disruption and/or disturbance to on-going site
activities.
4. Time required for subsurface remediation may be shorter than
other approaches (e.g., pump-and-treat).
5. Generally recognized as being less costly than other remedial
options.
6. Can be combined with other technologies (e.g., bioventing, soil
vapor extraction).
7. Usually does not produce waste products requiring disposal.

121. disadvantages of engineered in situ
bioremediation
1. Injection wells and/or infiltration galleries may become
plugged
by microbial growth or mineral precipitation.
2. High concentrations (TPH > 50,000 ppm) of low solubility
constituents may be toxic and/or not bioavailable.
3. Difficult to implement in low-permeability aquifers.
4. Re-injection wells or infiltration galleries may require permits
or
may be prohibited (Some states require permit for air injection).
5. May require continuous monitoring and maintenance.
6. Remediation may only occur in a more permeable layer or
channels within the aquifer.

122. advantages of intrinsic bioremediation
1. Lower costs than most active remedial alternatives.
2. Minimal disturbance to the site operations.

123. disadvantages of intrinsic bioremediation
1. Not effective where contaminant concentrations are high
(e.g., > 20,000 to 25,000 ppm TPH).
2. Not suitable under certain site conditions (e.g., impacted
ground water supply, presence of free products).
3. Some migration of contaminants may occur; not suitable if
receptors might be affected.
4. Long period of time required to remediate relatively
recalcitrant
contaminants.
5. Longer period of time may be required to mitigate
contamination than for active remedial measures.
6. May not always achieve the desired cleanup levels within a
reasonable length of time.

125. Two main types of treatment walls
Permeable reactive trench:
this is the simplest form of treatment walls and it consists of
a trench that extends across the entire width of the plume.
The system is installed by digging a trench and filling it with
Permeable material. As the contaminant plume moves
through the wall, contaminants are removed by various mass
Transfer processes such as air stripping, SVE, and adsorption

126. Permeable reactive barrier. (Adapted from EPA, 2000)

127. Funnel and gate systems:
Used primarily when contaminated plumes are too large or
too deep to dig a trench across its width.
When dealing with funnel and gate systems, the gate is used
to pass contaminated groundwater through the reactive wall,
and the funnel is integrated into the system to force water
through its gates.

129. Treatment options
 Treatments walls are often used for groundwater
contaminated with VOCs, SVOCs, and inorganics.
 This technology is ineffective in treating other fuel
hydrocarbons

130. limitations
 It is limited to a subsurface lithology that has a
continuous aquitard at a depth that is within the vertical
limits of the trenching equipment.
 Passive treatment walls have a tendency to lose their
reactive capacity over time, and require replacement of
the reactive medium.
 Large and deep plumes are more difficult to remediate
than small and shallow plumes.

132. Bioremediation Consortium

136. Ex-situ - Bioslurry systems
Ex situ biological treatment requires excavation of
contaminated soil. It is accomplished by combining
the excavated soil with water and other additives.
In this System the bacteria selected for breaking down
the contaminant is also added. The excavated soil is
treated in a controlled bioreactor where the slurry is
mixed to keep the solids suspended and the
microorganisms in contact with the contaminants.

137. Ex-situ slurry-phase bioremediation

138. Bioslurry/bioreactors are successful in treating
Nonhalogenated SVOCs and VOCs in excavated soils
Or dredged sediments.
Other contaminants include ordinance
compounds, pesticides, and PCBs.

139. performance of bioslurry technology are:
 Bioslurry/bioreactors are technically simple, and pellet
formation can be avoided during dry treatment.
 It works on most petroleum types.
 It is relatively simple and versatile.
 It is more effective than bioremediation.
 Closed systems allow the control of temperature,
moisture, pH, oxygen, nutrients, addition of surfactants,
supplementation of microorganisms, monitoring of
reactions and conditions, and the control of VOC
emissions.

140. Limitations
 Excavation is required.
 Non-homogeneous and clayey soils can cause serious
handling problems.
 In the case of free product, removal is necessary.
 Dewatering of fine soils after treatment can be expensive.
 A disposal method is needed for non-recycled
 wastewater.
 It may require extensive site and contaminant
characterization (chemical reactivity, vapor pressure,
biodegradability,etc.).

141. lagoons

142. Contained ex-situ solid phase bioremediation

144. BIOREMEDIATION OF FRESH
WATER AND MARINE
SYSTEMS

146. Oil spill!!
 As a result of the petroleum industry millions of tons of oil
enter the oceans every year
 Accidental releases may contribute only a small
percentage of the oil released into the marine environment
 Accidental oil spills receive much attention and public
concern
 They can result in contamination of ocean and shoreline
environments

147.  The biggest spill ever occurred during the 1991 Persian Gulf
war - about 240 million gallons of oil spilled off the coast of
Prince William Sound, Alaska
 The Exxon Valdez accident at Bligh Reef in 1989 discharged
40 million litres….
.

148. EFFECTS
 Prevent the dissolution of oxygen to water
 Marine mammals and birds are highly affected
 Coastal and inter tidal flora and fauna are damaged

149. The Fate of Oil in the Marine Environment
Abiological weathering processes include
 Evaporation
 Dissolution,
 Dispersion
 Photochemical oxidation,
 Water-in-oil emulsification,
 Adsorption onto suspended particulate material, sinking
 Sedimentation

150. Biological processes include
 Ingestion by organisms as well as microbial degradation
 In general, it is the process where by microorganisms chemically
transform compounds such as petroleum hydrocarbons into
simpler products

151. CLEAN UP TECHNOLOGIES
 Physical/Mechanical
 Chemical
 Biological (bioremediation)

154. What is Bioremediation?
 Microbes degrading toxic substances
Requirements
 Identification of microbes capable of degrading petroleum
hydrocarbons.
 Nutrient requirements of these microbes, such as carbon,
nitrogen and phosphorous.
 Environmental requirements such as oxygen, water and
temperature.
 Metabolic pathways of decomposition for oil fractions

155. Biodegradation of oil
 Crude oil is a complex mixture of thousands of different chemical
compounds
 No crude oil is subject to complete biodegradation
 All marine and freshwater ecosystems contain some oil-
degrading bacteria
 No one species of micro-organism, is capable of degrading all
the components of oil
 Different species are required for significant overall degradation

156.  Hydrocarbon-degrading bacteria make up
less than 1 percent of the bacterial population
 In most chronically polluted system they constitute 10 percent or
more of the total population
 These compounds are a rich source of the
carbon and energy that microbes require for growth

157.  The activity of microorganisms at a spill site is related to it’s
ability to produce enzymes to catalyze metabolic reactions.
 These different enzymes and metabolic pathways, cannot be
found in any single species

158. On-duty

159. MICROORGANISMS
Bacteria Fungi
Achrornobacter Aspergillus
Acinetobacter Candida
Actinomyces Mucor
Aeromonas Penicillium
Alcaligenes Saccharomyces
Arthrobacter Rhodosporidium
Klebsiella
Lactobacillus

160. Metabolic Pathways
 Numerous and varied
 Four components to oil
 Saturated hydrocarbons, aromatic hydrocarbons, resins, and
asphaltenes
 Pathways for asphaltenes are not understood, resins are only
biodegradable in small amounts

161. Aliphatics
 Also known as the saturates
 Includes compound such as n-paraffins, iso-paraffins and
alicyclic hydrocarbons (cycloparaffins)
 The straight-chain alkane compounds with 10 to 24 carbon
atoms are degraded the fastest
 As the length and branching increase degradability decreases

162. Aromatics
 Include monocyclic (benzene, toluene and xylene) poly cyclic
aromatics(naphthalene, anthracene and phenanthrene)
 These compounds can be degradable when they are simple and
have a low molecular weight
 Aromatics with five or more rings are persistant for long periods
of time

163. Asphaltenes
 They are complex and of high molecular weight
 Difficult to analyze with current methodology
 Tar is rich with asphaltenes
 Non biodegradable or slowly degradable

164. Resins
 Include petrolium compounds containing nitrogen, sulfur or
oxygen
 About 20% of heavy oil
 Limitted microbial degradation

165. Environmental Influences on
Biodegradation
Oxygen
 Its availability is rarely a rate-limiting factor in the
biodegradation of marine oil spills
 Microorganisms employ oxygen-incorporating enzymes to
initiate attack on hydrocarbons
 When oxygen is less available,the rates of biodegradation
decreases
 Oxygen availability is determined by depth in the sediment,
height of the water column, and turbulence

166. Nutrients
 Nutrients such as nitrogen, phosphorus, and iron play critical
role in biodegradation in marine waters
 Inadequate supply of these nutrients may result in a slow rate of
biodegradation
 Petroleum is deficient in the mineral nutrients to support
microbial growth
 phosphorus precipitates as calcium phosphate at the pH of
seawater

167. Temperature
 The temperature of most seawater is between–2 and 35 degree C
 The rates of biodegradation are fastest at the higher end of this
range and decrease in very cold climates
 A temperature drop from 25 to 5 degree C caused a tenfold
decrease in response
 Oil becomes more viscous at low temperature-less
spreading occurs and less surface area is available for
colonization by microorganisms.

168. Pressure
 Increasing pressure decreases the rates of
biodegradation
 Pressure is very important in the deep ocean

169. Salinity
 Microorganisms in oceans are typically well adapted with the
range of salinities
 Not very important in marine environment

170. pH
 Extremes in pH affect a microbe’s ability to degrade
hydrocarbons.
 pH does not fluctuate much in the oceans-it remains between 7.6
and 8. l
 Do not have an important effect on biodegradation rates in most
marine environments.

171. concentration
 Concentration of pollutant is important
 If concentration is very high it reduces the amount of
oxygen,water and nutrients avaliable to microbes
 So microbes are stressed, reducing ability to degradation

172. steps
Pre-treatmant assessment
Evaluation of if bioremediation is a viable option based on
 Type of contaminant
 Its concentration
 Presence of potential microbial degraders
 Concentration of back ground nutrients
 Type of ecosystem

173. Rocky

174. Design of treatment and monitoring plan
 Further assessment and planning prior to the application
Involves
 Selection of rate limiting treatment agenda
 Determination of application o strategies
 Design of sampling and monitoring plans

175. Assessment and termination of process
 Assessment of treatment efficiency
 Determination of appropriate treatment end points using
chemical, toxicological and ecological analysis

176. TECHNOLOGIES
1) Nutrient enrichment
 To overcome the chief limitation on the rate of the
natural biodegradation of oil
 Nutrients are added to spill site that limit
biodegradation rates
 Nutrient enrichment promotes bioremediation

177. 2) seeding with naturally occurring
microorganisms
 Addition of microorganisms to promote increased rates of
biodegradation
 The inoculum may be
- a blend of nonindigenous microbes from various polluted
environments
-a mix of oil-degrading microbes selected from the site to
be remediated and mass-cultured in the laboratory

178. 3) seeding with genetically engineered
microorganisms (GEMs)
 Genetically modified microorganisms with high rate of oil
degradation are added
 More efficient than naturally occurring species
 Degrade fractions of petroleum not degradable by naturally
occurring species
 GEMs are not usually appiled due to the increased
problems associated with GEMs

179. Advantages
 Minimal physical disruption of a site
 No significant adverse effects
 Helpful in removing some of the toxic components of oil
 Simpler and more Possibly less costly than other approaches

180. Disadvantages
 Undetermined effectiveness for many types of spills
 Takes time to work
 Approach must be specific for each polluted site
 Optimization requires substantial information about spill site
 The possible toxicity of fertilizer components may cause health
problems
 Introduced organisms may be pathogenic to other life forms
 GEMs may have cetain potential impacts on that environment

181. Summary